The safe and efficient delivery of DNA to cells presents a formidable challenge and an obstacle to the clinical success of gene therapy. Anderson, W. F. (1998) Human Gene Therapy. Nature, 392 Suppl. 25-30; Verma, I. M.; Somia, N. (1997) Gene Therapy—Promises, Problems, and Prospects. Nature, 389, 239-242; Crystal, R. G. (1995) Transfer of Genes to Humans: Early Lessons and Obstacles to Success. Science, 270, 404-410. Synthetic polymers have been investigated widely as gene delivery agents and are generally viewed as long-term alternatives to viruses due to their low immunogenicities and the ease with which they can be structurally modified. Luo, D.; Saltzman, W. M. (2000) Synthetic DNA Delivery Systems. Nat. Biotechnol., 18, 33-37. Cationic polymers are particularly useful in this context because they form conjugates with negatively charged DNA, and the incorporation of new design elements into cationic polymers has resulted in advances toward functional gene delivery systems. Despite extensive work, however, polymers remain far less efficient than their viral counterparts.
For efficient gene transfer and expression to occur, a gene delivery agent (or vector) should overcome numerous intracellular barriers to transfection. Luo, D.; Saltzman, W. M. (2000) Synthetic DNA Delivery Systems. Nat. Biotechnol., 18, 33-37. For example, a vector should be able to: 1) condense DNA into stabilized, nanometer-scale structures, 2) target cells and stimulate internalization, 3) prevent the degradation of DNA inside the cell, 4) target the cell nucleus, and 5) release DNA in the nucleus so that it is available for transcription. Progress has been made toward many of these barriers—cationic polymers are used to condense DNA into 50 to 200 nm particles (barrier 1), conjugation with cell-specific ligands can be used to target complexes and stimulate uptake (barrier 2), the incorporation of pH-buffering functionality into synthetic polymers provides protection against acidic intracellular environments (barrier 3), and the nuclear membrane has been “breached” (barrier 4). Kabanov, A. V.; Felgner, P. L.; Seymour, L. W., in Self-Assembling Complexes for Gene Delivery: From Laboratory to Clinical Trial, John Wiley and Sons, New York, 1998; Putnam, D.; Gentry, C. A.; Pack, D. W.; Langer, R. (2001) Polymer-Based Gene delivery with Low Cytotoxicity By a Unique Balance of Side-Chain Termini. Proc. Natl. Acad. Sci. USA, 98, 1200-1205; Midoux, P.; Monsigny, M. (1999) Efficient Gene Transfer by Histidylated Polylysine/pDNA Complexes. Bioconjugate Chem., 10, 406-411; Boussif, O.; Lezoualc' H, F.; Zanta, M. A.; Mergny, M. D.; Scherman, D.; Demeneix, B.; Behr, J. P. (1995) A Versatile vector for Gene and Oligonucleotide Transfer Into Cells in Culture and In Vivo—Polyethyleneimine Proc. Natl. Acad. Sci. USA, 92, 7297-7301; Benns, J. M.; Choi, J.; Mahato, R. I.; Park, J.; Kim, S. W. (2000) pH-sensitive Cationic Polymer Gene Delivery Vehicle: N-Ac-poly(L-histidine)-graft-poly(L-lysine) Comb Shaped Polymer. Bioconjugate Chem., 11, 67-645; Wolff, J. A.; Sebestyén, M. G. (2001) Nuclear Security Breached. Nature Biotechnol., 19, 1118-1120; Rebuffat, A.; Bernasconi, A.; Ceppi, M.; Wehrli, H.; Verca, S. B.; Ibrahim, M.; Frey, B. M.; Frey, F. J.; Rusconi, S. (2001) Selective Enhancement of Gene transfer by Steroid-Mediated Gene Delivery. Nature Biotechnol., 9, 1155-1161. These recent successes have fueled hopes of a “grand design” in which individual design elements could be assembled to create synthetic vectors that functionally mimic viruses. Wolff, J. A. (2002) The “Grand” Problem of Synthetic Delivery. Nature Biotechnol., 20, 768-769. However, the breach of early barriers to transfection simply places increased significance on downstream barriers, and the design of materials to address the fifth and final barrier—the efficient and timely separation of polymer from DNA in the nucleus—has not been adequately addressed.
This “ultimate” barrier to efficient transfection presents a challenging problem from a design perspective, as designing methods to surmount it can introduce a functionality that is contrary to that required for efficient DNA condensation (i.e., barrier 1). Kircheis, R.; Wightman, L.; Wagner, E. (2001) Design and Gene Delivery Activity of Modified Polyethyleneimines. Advanced Drug Delivery Reviews, 53, 341-358. Cationic polymers spontaneously self-assemble with anionic DNA through electrostatic interactions to form condensed interpolyelectrolyte complexes—a process that is driven entropically by the elimination of small salts (e.g., NaCl) formed upon complex formation. Kabanov, A. V.; Feigner, P. L.; Seymour, L. W., in Self-Assembling Complexes for Gene Delivery: From Laboratory to Clinical Trial, John Wiley and Sons, New York, 1998.
Cationic polymers undergo self-assembly with anionic plasmid DNA to form condensed complexes. The reverse of this process—the intracellular dissociation of DNA from condensed interpolyelectrolyte complexes—appears to be unfavorable under physiological conditions and presents a substantial obstacle to efficient gene delivery.
Although the effects of pH, temperature, salt concentration, and molecular weight on the dissociation of model interpolyelectrolyte complexes are generally well understood, the mechanisms through which dissociation occurs for polymer complexes in the cytoplasm or nucleus of a cell are currently unclear. Bronich, T. K.; Nguyen, H. K.; Eisenberg, A.; Kabanov, A. V. (2000) Recognition of DNA Topology in Reactions Between Plasmid DNA and Cationic Polymers. J. Am. Chem. Soc., 122, 8339-8343. That meaningful levels of transfection are observed using polymeric vectors suggests that dissociation does occur, presumably mediated by ion exchange with other intracellular polyelectrolytes. However, recent analytical experiments suggest that DNA/polycation complexes are stable toward intracellular dissociation and that the inefficiency of this “unpackaging” process presents a substantial physical barrier to transfection. Godbey, W. T.; Wu, K.; Mikos, A. G. (1999) Tracking the Intracellular Path of Poly(ethyleneimine)/DNA Complexes for Gene Delivery. Proc. Natl. Acad. Sci. USA, 96, 5177-5181; Schaffer, D. V.; Fidelman, N. A.; Dan, N.; Lauffenburger, D. A. (2000) Vector Unpackaging as a Potential Barrier for Receptor-Mediated Polyplex Gene Delivery. Biotechnol. Bioeng., 67, 598-606.